Quantitative analyses of pharmaceutically and biologically important compounds, such as drugs and metabolites, are important applications of mass spectroscopy. Traditionally, ion sources based on electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are used in combination with triple-quadrupole mass spectrometers (triple quads) to provide quantitative analysis. The combination provides both high sensitivity and high specificity. ESI and APCI both generate ions from flowing liquid streams, and are therefore used by pumping organic and aqueous solvent streams containing the compounds to be analyzed through the source. Liquid chromatography is commonly used as an on-line separation technique prior to the mass spectrometer. Thus, samples can be introduced by injecting a known volume containing the sample into the liquid flow, and using the mass spectrometer to monitor specific combinations of ion mass/charge values that correspond to known precursor and product fragment ions using the scan mode known as multiple-reaction-monitoring (MRM) mode. During the scan, samples are injected sequentially, at a rate in the order of 1 per 10 second, due to limitations in autosamplers, as well as limitations imposed by the natural width of the eluting peak. Once the sample has passed through the ion source, it is ionized and dissipated in the source, with only a small fraction of the ions generated from the sample actually being sampled into the mass spectrometer system.
Matrix assisted laser desorption/time-of-flight (MALDI/TOF) is a different type of mass spectrometer technique, in which samples are mixed with a UV-absorbing compound (the matrix), deposited on a surface, and then ionized with fast laser pulses. A short burst or plume of ions is created in the ion source of the mass spectrometer by the laser, and this plume of ions is analyzed by a time-of-flight mass spectrometer, by measuring the flight time over a fixed distance (starting with the ion creating pulse). This technique is inherently a pulsed ionization technique (required for the time-of-flight mass spectrometer) as well as a batch-processing technique, since samples are introduced into the ion source in a batch (of samples located in small spots on a plate) rather than in a continuous flowing liquid stream. MALDI/TOF has been almost exclusively used for the analysis of biopolymers such as peptides and proteins. The technique is sensitive and works well for fragile molecules such as those mentioned, and the TOF method is particularly suitable for the analysis of high-mass compounds. However, until recently, there has been no viable method of doing true MS/MS with this type of instrument. Instead, the method of post-source decay (PSD) is used to provide some fragmentation information. In this technique, precursor ions are selected in the flight tube with an ion gate, and then those ions that fragment before the ion mirror (due to excess energy carried away from the source) can be mass resolved. This technique provides relatively poor sensitivity and mass accuracy, and is not considered to be a high performance MS/MS technique. The MALDI technique also suffers from the fact that while the mass accuracy and resolution can be very high (up to 30,000 resolution at low mass, and accuracy of a few parts-per-million), these important features are difficult to achieve because they depend on the microstructure of the sample surface (roughness), the laser fluence, and other instrumental characteristics which can be hard to control. Good mass accuracy typically requires that calibration compounds be placed on the sample surface close to the actual sample itself. The MALDI/TOF technique has mainly been used for spectral analyses. Some previous attempts have been made to use MALDI for quantitative analysis, but they have met with limited success because of the poor precision obtained with MALDI/TOF.
Recently, the method of combining MALDI with orthogonal TOF has been introduced by a group at the University of Manitoba. This technique, called Orthogonal MALDI, or “oMALDI™” (trademark of Applied Biosystems/MDS SCIEX Instruments, Concord, Ontario, Canada) as described in U.S. Pat. No. 6,331,702 (assigned to the University of Manitoba), is an apparatus and method enabling a pulsed source, such as a MALDI source, to be coupled to a variety of spectrometer instruments, in a manner which more completely decouples the spectrometer from the source and provides a more continuous ion beam with smaller angular and velocity spreads. In this technique, ions generated from a MALDI source as plumes (typically at the rate of less than 20 Hz, with pulse widths of a few nanoseconds from the laser pulse) are collisionally cooled in a relatively high pressure region containing a damping gas within an RF ion guide. Collisions with the damping gas convert the plumes into a quasi-continuous beam. This quasi-continuous beam is then analyzed with orthogonal time-of-flight, in which the ions enter orthogonally to the axis of the TOF and are pulsed radially.
There are several advantages to this combination that are not available from conventional MALDI/TOF. The TOF resolution and mass accuracy are decoupled from the source conditions such as laser fluence and sample morphology. The ions are slowed to near thermal energies from which they can conveniently be re-accelerated to tens of electron volts for collisionally activated decomposition (CAD) in a collision cell. The flux of ions in the beam is low enough (through having the beam stretched out in time) that a time-to-digital converter (TDC) can be used for ion detection. The result is that high mass accuracy and resolution can be achieved under a wide range of operating conditions. In addition, a mass resolving quadrupole and collision cell can be placed before the TOF analyzer to provide an MS/MS configuration. Precursor ions from the MALDI source are collisionally cooled, then selected by the quadrupole mass filter, fragmented in the collision cell, and the fragments mass analyzed by the TOF. This provides high mass resolution and sensitivity for MS/MS of MALDI ions, which has not been previously available. This MS/MS configuration is referred to as QqTOF, where Q refers to the mass filter quadrupole and q refers to the RF-only collision cell.
The Manitoba group recognized that the oMALDI™ technique allows a MALDI source to be efficiently coupled to a quadrupole mass spectrometer system, because of the near-continuous nature of the ion beam. However, there is no recognition that this might offer improved ability to measure sample concentrations quantitatively.
One important factor for certain applications regarding the performance of any ion mass analyzing technique is the ability to effectively capture ions from the ion source and transport them to the analyzing device so that they can be analyzed. This factor is of particular importance for analytical techniques, such as the MRM scan, that are directed to quantitative measurements of ions of interest. With a MALDI source, each laser pulse impinging on the MALDI target generates a plume of ions and neutral particles. The ions have to be delivered to a mass analyzer, either directly or through an ion transport device, so that they can be analyzed. The mass analyzer or ion transport device, such as a quadrupole ion guide, typically has a very limited ion acceptance zone, and an inlet aperture is often used to collimate the incoming ions. An aperture is also often used for the purposes of preventing unwanted materials released by the sample target from contaminating the mass analyzer or ion guide, and separating the MALDI source from the downstream vacuum chamber in which the mass analyzer or ion transport device is located. Such an aperture is typically significantly smaller than the width of the plume of ions coming off the MALDI target. As a result, a significant portion of the ions in the plume cannot enter the aperture and be detected by the mass analyzer. Also, the central axis of the plume may be at an angle from the aperture, which further reduces the portion of ions that will go through the aperture. As a result, the measurement sensitivity of the mass spectrometer is significantly reduced, as the sampling efficiency of the ions, i.e., the portion of ions from the ion source that is analyzed by the downstream mass analyzer, can be rather low.